1. Field of the Invention
This invention relates to novel fluorescent tagging agents. More specifically, this invention relates to novel fluorescent tagging agents capable of tagging oilgosaccharides to form oligosaccharide-biotin adducts useful in analytical, diagnostic, and therapeutic application.
2. Description of Related Art
Many of the important biological roles of oligosaccharides involve their interaction with specific receptor proteins. Studies of these interactions require specific oligosaccharides from various biological sources. Isolation of oligosaccharides in individually, structurally definable units is critical to such studies. For the fractionation and detection of oligosaccharides, currently available methods include metabolic labelling, chemical labelling, and high pH anion exchange chromatography with pulse-amperometric detection, all of which are fraught with some problems.
An alternative approach is the tagging of free oligosaccharides by reductive amination with amino-phospholipids or with neutral or acidic fluorescent reagents. Although this approach is useful and practical in some applications, fractionation of the tagged oligosaccharides has certain limitations. Additionally, the fractionated, purified oligosaccharides so obtained are of limited use for purposes other than structural analysis. The only exception is phospholipid tagging, which allows probing of TLC plates by purified receptors that are already known to recognize oligosaccharides. However, these lipid adducts cannot be used as soluble reagents to probe natural or recombinant sources for previously unknown oligosaccharide receptors. Several alternative approaches are available for the probing of oligosaccharide receptors. Neoglycoproteins can be prepared by coupling of defined, unmodified oligosaccharides to proteins such as bovine serum albumin. Neoglycoproteins are used as probe tools with target specificity to cells. Also, oligosaccharides or glycopeptides can be coupled to biotin, resulting in the formation of tight complexes with avidin or streptavidin. The latter approach can utilize the wellknown avidin-biotin technology (Wilcheck, M.; Bayer, E. A., Anal. Biochem. 1988, 171, 1-32). However, both approaches suffer from some drawbacks: the neoglycoprotein approach is limited by heterogenous stoichiometry and arrangement of the coupled oligosaccharides on the protein which cannot be controlled; and the biotin approach requires that the oligosaccharide or glycopeptide be first purified to homogeneity, prior to coupling.
In the area of immunology, it has always been problematic to produce a meaningful immune response to carbohydrate antigens. Typically, the immune response which does occur is in the form of a low-affinity IgM, probably due to the fact that antibody responses to sugar chains are T-cell dependent. In addition to low affinity, the IgM produced in response to carbohydrate antigens is often subject to non-specific cross-reactivity with related compounds. In trying to produce a higher affinity IgG response to carbohydrate antigens, researchers have typically resorted to covalent coupling of the carbohydrate antigen to a carrier, such as a foreign protein. However, this latter technique is somewhat laborious and often requires tedious absorption steps to remove antibodies produced in response to the carrier. Thus, considerable need exists for a routine technique which would allow immunogenic presentation of an individual oligosaccharide and purification of oligosaccharide specific immunoglobulin.
Accordingly, there is a long-felt need for a tagging agent for purposes of tagging and fractionating oligosaccharides which allows further application of the tagged, fractionated oligosaccharides. This invention disclosed herein enables the synthesis of novel fluorescent tagging compounds and fluorescent tagged oligosaccharide adducts that possess many desirable properties.